Clinical laboratory tests are routinely performed on the serum or plasma of whole blood. In a routine assay, red blood cells (RBC) are separated from plasma by centrifugation, or RBC's and various plasma proteins are separated from serum by clotting prior to centrifugation. Hb, light-scattering substances like lipid particles, and bile pigments bilirubin (BR) and biliverdin (BV) are typical blood components, which will interfere with and affect spectroscopic and other blood analytical measurements of blood analytes. Such components are referred to as interferents, and they can be measured by spectroscopic methods. The presence of such interferents affects the ability to perform tests on the serum or plasma and as such can be said to compromise sample integrity.
CO-oximetry is a technique used to measure the major Hb species in whole blood, for example, Oxy-Hb, Deoxy-Hb, Met-Hb, Carboxy-Hb, and Sulf-Hb. The technique of CO-oximetry requires that the whole blood is collected in such a manner as to protect the sample from exposure to air, so as to minimize conversion of Deoxy-Hb into Oxy-Hb. Further, the technique requires that the RBC's be hemolyzed to provide an optically transparent sample in the cuvette for measurement. The most commonly used method of hemolyzing a whole blood sample is to expose the sample to the sound waves of an ultrasonic sound generator; chemical hemolyzing agents may also be used. Current methods of CO-oximetry use the extinction coefficient of the Hb species at different wavelengths, in the calibration algorithms. They are not designed to deal with the light-scattering effect created by RBC's, hence the requirement of a hemolyzing system. Some CO-oximetry methods, for example U.S. Pat. No. 4,997,769, suggest that they can mathematically deal with scattering of light by small particles, for example chylomicrons (an RBC is 1-2 orders of magnitude larger than a chylomicron), but a hemolyzing system is still required.
Current methods of measuring Total-Hemoglobin (Tot-Hb) in a sample, preferably use reagents, whereby the different Hb species like Oxy-hemoglobin (Oxy-Hb), Deoxy-Hemoglobin (Deoxy-Hb), Carboxy-Hemoglobin (Carboxy-Hb), and Met-Hemoglobin (Met-Hb) are converted to a single specie, which is then measured at one wavelength using spectroscopic methods; sometimes a second wavelength is also used. The reagents are usually noxious (e.g. potassium cyanide and azide), and there is a need for a reagentless method for measuring Hb in body fluids. Harboe (Harboe, M., 1959, A method of determination of hemoglobin in plasma by near ultraviolet spectrophotometry. Scand. J. Clin. Lab. Invest, pp. 66-70) and Tietz (Tietz Textbook of Clinical Chemistry, 3rd Ed, 1999, pp 1674-1676; which is incorporated herein by reference), provide examples of reagentless spectroscopic methods for measuring Hb. Although Hb provides very large absorbance signals, the absorbance spectra of the Hb species exhibit significant differences. Reagentless spectroscopic methods are limited to samples that contain mostly Oxy-Hb and Deoxy-Hb. The Deoxy-Hb is usually converted into Oxy-Hb when the sample is exposed briefly to atmospheric oxygen. The largest source of errors in both methods (Harboe & Tietz) is the presence of Met-Hb. In U.S. Pat. No. 6,689,612 (Samsoondar), there is described the use of Total-Hb, Oxy-Hemoglobin (Oxy-Hb), and “Total-Hb minus Met-Hemoglobin (Met-Hb),” as indicators of hemolysis. Because the absorbance spectrum for Met-Hb is so different from the other Hb species, a calibration algorithm developed for Hb may be better at predicting “Total-Hb minus Met-Hb.”
Met-Hb is an oxidation product of Hb and the Met-Hb form of the Hemoglobin-based (Hb-based) blood substitutes is also an oxidation product of Hemoglobin-based blood substitutes. Met-Hb from natural Hb, or Hb-based blood substitutes, cannot carry oxygen, and therefore Met-Hb, or Met-Hb form of the Hemoglobin-based blood substitutes are not functional Hb.